Re: 발효과정에서 DHA, EPA 생산가능한가?

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곡물을 발효할 때, 미생물이 생존하면서 다양한 대사산물을 분비하게 됩니다. 이는 미생물의 종류와 발효 조건에 따라 달라질 수 있지만, 일반적으로 다음과 같은 물질들이 생성됩니다.

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1. 유기산(Organic Acids)

미생물이 곡물 속의 탄수화물을 분해하면서 젖산, 초산, 구연산, 푸마르산 등의 유기산을 생성함.

• 젖산(Lactic acid): 젖산균(락토바실러스)이 생성, pH를 낮춰 부패 방지.
• 초산(Acetic acid): 초산균(Acetobacter)이 생성, 신맛과 방부 효과.
• 푸마르산(Fumaric acid): 일부 곰팡이(예: Rhizopus)에서 생성.

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2. 알코올류(Alcohols)

곡물 속의 당이 발효되면서 효모(Saccharomyces)나 일부 곰팡이가 에탄올, 부탄올, 글리세롤 등을 생성함.

• 에탄올(Ethanol): 효모 발효 시 주로 생성되며, 술(막걸리, 청주 등)의 주요 성분.
• 부탄올(Butanol): 클로스트리디움(Clostridium) 균에 의해 생성될 수 있음.
• 글리세롤(Glycerol): 효모가 생존을 위해 일부 생성하는 물질.

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3. 지질(기능성 지방산, Lipids & Fatty Acids)

일부 **곰팡이(Mucor, Mortierella, Rhizopus 등)**는 곡물의 탄수화물을 지질로 전환하여 저장함.

• 감마 리놀렌산(GLA, Gamma-Linolenic Acid): Mucor circinelloides가 생산 가능.
• 아라키돈산(AA, Arachidonic Acid): Mortierella속 곰팡이가 생산.
• DHA, EPA: 일부 미세조류가 생산.

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4. 폴리페놀 대사산물(Polyphenol Derivatives)

곡물 속의 폴리페놀(polyphenol) 성분이 미생물에 의해 변형됨.

• 카테킨(Catechin) 변환: 녹차 발효 시 테아플라빈(홍차 성분) 형성.
• 이소플라본(Isopflavone) 변화: 대두 발효 시 다이드제인, 제니스테인 증가.

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5. 단백질 및 펩타이드(Proteins & Peptides)

곡물 속 단백질이 미생물 효소(프로테아제)에 의해 분해되어 아미노산, 펩타이드가 생성됨.

• 감칠맛 펩타이드(Umami Peptides): 발효 간장에서 생성됨.
• 생리활성 펩타이드(Bioactive Peptides): 혈압 강하 효과 등.

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6. 기타 기능성 대사산물

• 폴리올(Polyols): 일부 효모와 곰팡이가 소르비톨, 만니톨 같은 당알코올을 생성.
• 멜라노이딘(Melanoidin): 갈변 반응으로 생성, 항산화 효과.
• 비타민(Vitamins): 비타민 B군(특히 B12)이 일부 발효균에서 생성됨.

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결론

곡물 발효 과정에서 미생물이 생존하면서 다양한 대사산물을 분비하며, 이는 발효식품의 풍미, 기능성, 보존성에 영향을 미침.
특정 미생물을 활용하면 기능성 지질(GLA, DHA 등)이나 생리활성 펩타이드를 의도적으로 증가시킬 수도 있음.

곡물 발효 과정에서 박테리아에 의해 생성되는 DHA와 EPA

DHA(도코사헥사엔산, Docosahexaenoic acid)와 EPA(에이코사펜타엔산, Eicosapentaenoic acid)는 오메가-3 지방산으로, 주로 해양 미세조류(microalgae)나 일부 박테리아에 의해 생성됩니다. 곡물 발효 과정에서 특정 미생물이 존재하면 DHA와 EPA가 생산될 가능성이 있습니다.

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1. DHA 및 EPA를 생산하는 박테리아

곡물 발효 과정에서 DHA와 EPA를 생산할 수 있는 주요 미생물은 다음과 같습니다.

(1) 해양성 박테리아(Marine Bacteria)

• 모르텔렐라속 (Mortierella)
• 슈도모나스속 (Pseudomonas)
• 쉐와넬라속 (Shewanella)
• 알테로모나스속 (Alteromonas)
• 페디오코쿠스속 (Pediococcus) (일부 종)

이들은 특정 환경에서 DHA와 EPA를 생산할 수 있습니다. 하지만 전통적인 곡물 발효 과정에서 자연적으로 발견되기는 어렵습니다.

(2) 오메가-3 생성 가능한 균주를 이용한 발효

일반적으로 곡물 발효에서 발견되는 균주는 젖산균(Lactobacillus), 효모(Saccharomyces), **곰팡이(Aspergillus, Rhizopus)**가 많으며, 이들 대부분은 DHA와 EPA를 직접 생성하지 않습니다.
그러나 특정 균주를 인위적으로 접종하면 곡물 발효 중 DHA와 EPA를 얻을 수 있습니다.

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2. DHA와 EPA 생성 조건

DHA와 EPA를 효과적으로 생성하려면 다음 조건이 필요합니다.

• 적절한 박테리아 접종: 자연 발효보다는 특정 DHA/EPA 생산 균주를 추가해야 함.
• 지질 전구체(lipid precursor) 공급: DHA/EPA 합성을 위해 불포화 지방산 공급 필요(예: 리놀렌산, α-리놀렌산).
• 온도 및 산소 조절: 일반적으로 20~30℃에서 DHA/EPA 생산이 활발함.
• 장기 발효: 단기간 발효보다는 장기간(수일~수주) 발효할 때 생성량 증가 가능.

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3. 곡물 발효에서 DHA/EPA를 얻는 방법

곡물 발효 중 자연적으로 DHA/EPA를 얻기 어렵지만, 다음 방법을 활용하면 가능성이 있습니다.

1. DHA/EPA 생산 미생물(예: Schizochytrium sp., Thraustochytrium sp.)을 곡물 발효 배지에 접종
2. 발효 배양액에 리놀렌산(α-Linolenic acid) 같은 전구체 추가
3. 산소 농도를 조절하여 미생물의 지질 합성 경로 유도

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4. 결론

곡물 발효 과정에서 일반적인 박테리아는 DHA와 EPA를 직접 생산하지 않지만, 특정 미생물을 접종하고 적절한 환경을 조성하면 DHA/EPA 생산이 가능합니다. 특히 Schizochytrium, Thraustochytrium 같은 해양성 미생물을 활용하면 곡물 발효를 통한 오메가-3 지방산 생산이 가능할 수 있습니다.

1. nature  
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4. article

Lipid production from corn straw by cellobiohydrolase and delta-6 desaturase engineered Mucor circinelloides strains under solid state fermentation

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• Published: 13 August 2024

Lipid production from corn straw by cellobiohydrolase and delta-6 desaturase engineered Mucor circinelloides strains under solid state fermentation

• Yao Zhang, 
• Zhuo Liu, 
• Yan Sun, 
• Yuanxin Du, 
• Zixuan Zhao, 
• Qing Liu & 
• Yuanda Song 

Scientific Reports volume 14, Article number: 18784 (2024) Cite this article

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Abstract

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry.

이전에는 셀룰로오스를 활용하는 것뿐만 아니라

γ-리놀렌산(GLA)의 생산량을 증가시킬 수 있는

M. circinelloides 변종을 만들었습니다.

이번 연구에서는 옥수수 짚을 사용하여 M. circinelloides 변종에 의한 지질 축적에 대한 심층 분석을 탐구했습니다. 2단계 온도 조절 전략을 채택하여 셀룰라아제 1.5%와 접종물 15%를 추가했을 때, 조작된 균주는 대조 균주에 비해 지질 생산량(건조 배지 100g당 최대 1.56g)과 GLA 생산량(건조 배지 100g당 최대 274mg)이 각각 1.8배와 2.3배 증가했습니다. 이 연구는 조작된 M. circinelloides 균주, 특히 Mc-C2PD6이 옥수수 짚을 사용하여 GLA를 생산하는 데 장점이 있음을 입증했습니다. 이 연구는 농업용 셀룰로오스 폐기물을 한 단계로 기능성 지질로 전환하는 데 대한 참고 자료를 제공했으며, 이는 생물 산업계의 지속 가능한 발전을 촉진하는 데 긍정적인 역할을 할 수 있습니다.

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Introduction

Oleaginous microorganism such as bacteria, mold, yeast, algae, etc. can accumulate and synthesize a large amount of lipids in cells using carbohydrates under certain conditions, and the microbial lipids become an important alternative raw material for biodiesel production1,2,3,4. The fatty acid composition of microbial lipids is similar to that of common vegetable oil, and some also contain rich polyunsaturated fatty acids, such as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), etc.5,6,7,8. Although the development prospects of microbial lipids are broad, high cost is still the main factor restricting their industrial production.

Cellulosic waste is one of the most abundant and cheapest renewable raw materials in nature2,9,10. For example, crop straw is the main agricultural cellulosic waste, which is mostly used as feed, fuel, and organic fertilizer11,12,13. Using crop straw to produce microbial lipids can effectively reduce the production cost and realize the sustainable development of agriculture1,14.

The oleaginous fungus Mucor circinelloides, as the first commercially lipid producing strain in the world, can synthesize high level of γ-linolenic acid (GLA) with various important physiological functions15,16,17. Previously, we presented a novel approach by co-expression‎ of cellobiohydrolase and delta-6 desaturase in M. circinelloides to facilitate the GLA production from cellulose in one step15,18. In the present study, an in-depth analysis of lipid production by recombinant M. circinelloides strains Mc-C2TD6 and Mc-C2PD6 using corn straw under solid state fermentation was to be explored. The optimal addition amount of the cellulase and the inoculum of engineered M. circinelloides strains by using corn straw for lipid production were investigated. In addition, we developed a two-stage temperature control strategy for lipid production from corn straw under solid state fermentation. Our study provides a certain foundation for the further application of the engineered M. circinelloides strains in industrial production of microbial lipids from crop straw.

소개

박테리아, 곰팡이, 효모, 조류 등과 같은 유성 미생물은

특정 조건에서 탄수화물을 사용하여 세포에 다량의 지질을 축적하고 합성할 수 있으며,

미생물 지질은 바이오디젤 생산에 중요한 대체 원료가 됩니다1,2,3,4.

미생물 지질의 지방산 조성은

일반적인 식물성 기름과 비슷하며,

일부는 아라키돈산(AA), 도코사헥사엔산(DHA), 에이코사펜타엔산(EPA) 등과 같은

고도불포화지방산을 풍부하게 함유하고 있습니다5,6,7,8.

미생물 지질의 개발 전망은 광범위하지만,

높은 비용이 여전히 산업 생산을 제한하는 주요 요인입니다.

셀룰로오스 폐기물은

자연에서 가장 풍부하고 가장 저렴한 재생 가능한 원료 중 하나입니다2,9,10.

예를 들어,

작물 짚은 농업에서 가장 많이 발생하는 셀룰로오스 폐기물이며,

주로 사료, 연료, 유기질 비료로 사용됩니다11,12,13.

작물 짚을 사용하여 미생물 지질을 생산하면

생산 비용을 효과적으로 줄이고 농업의 지속 가능한 발전을 실현할 수 있습니다1,14.

무코르 시르시넬로이데스라는

지질성 곰팡이는 상업적으로 지질을 생산하는 세계 최초의 균주로서,

다양한 중요한 생리 기능을 가진

높은 수준의 감마리놀렌산(GLA)을 합성할 수 있습니다15,16,17.

이전에는

M. circinelloides에서 셀룰로오스로부터 GLA 생산을 한 단계로 촉진하기 위해

셀로바이오하이드롤라제와 델타-6 불포화효소를 공발현하는

새로운 접근법을 제시했습니다15,18.

본 연구에서는

고체 발효 조건에서 옥수수 짚을 사용하는 재조합

M. circinelloides 균주 Mc-C2TD6과 Mc-C2PD6에 의한 지질 생산에 대한

심층 분석을 탐구하고자 했습니다.

옥수수 짚을 이용한 지질 생산을 위한

최적의 셀룰라아제 첨가량과 M. circinelloides 변종 접종량을 조사했습니다.

또한,

고체 발효 조건에서 옥수수 짚을 이용한 지질 생산을 위한

2단계 온도 조절 전략을 개발했습니다.

이 연구는

작물 짚에서 미생물 지질을 산업적으로 생산하는 데

M. circinelloides 변종을 적용하는 데 필요한 기초를 제공합니다.

Materials and methods

Strains and media

The original M. circinelloides strain WJ11 and recombinant strains Mc-2075 (as a control, with empty plasmid), Mc-C2TD6 and Mc-C2PD6 (cellobiohydrolase and delta-6 desaturase co-expression‎) were previously constructed and stocked in our lab. The strains used in this work are summarized in Table 115,19.

Table 1 Strains used in this work.

Full size table

The seed medium is composed of glucose 20 g/L, peptone 20 g/L and yeast extract 10 g/L. The basic nutrient solution consists of: glucose 5 g/L, ammonium tartrate 2 g/L, KH2PO4 7 g/L, Na2HPO4 2 g/L, MgSO4·7H2O 1.5 g/L, yeast 1.5 g/L, CaCl2·2H2O 0.1 g/L, FeCl3·6H2O 8 mg/L, ZnSO4·7H2O 1 mg/L, CuSO4·5H2O 0.1 mg/L, Co(NO3)2·6H2O 0.1 mg/L, and MnSO4·5H2O 0.1 mg/L. The cellulase (5000 IFPU/g) is produced by Jinan Sansheng Biological Products Co., LTD.

The corn straw was collected from straw recycling center of Dongping, Tai’an, Shandong Province. The corn straw was baked in the oven at 80 °C until the quality remained unchanged, crushed, sifted 40 mesh for use. The corn straw solid fermentation medium is prepared by adding 80 g corn straw powder into 100 mL basic nutrient solution and then sterilizing.

Culture conditions

The recombinant strains of M. circinelloides stored in glycerol tube at – 80 ℃ were added into the seed medium and cultured in a shaker with a controlled rotational speed of 200 rpm/min, culture temperature of 28 °C, initial pH value of 5.0, and culture time of 24 h to obtain the seed liquid.

The corn straw solid fermentation medium was prepared and sterilized by high-pressure steam at 121 °C for 20 min. And then 0.0–2.0% (w/w) cellulase powder (protein content was 1 mg/mL) and above cultured seed liquid was added at 5.0–20.0% (w/w) of the inoculated amount. The fermentation culture was placed in a constant temperature incubator at 28 °C, pH 4.5 for 192 h. The two-stage temperature control strategy was 32 °C for the first 48 h and 28 °C for the last 144 h. It should be noted that among the above fermentation parameters, the temperature, the addition amount of cellulase and the inoculum of strains depend on the experimental design. All experiments were carried out in triplicate.

Determination of lipid yield

The fermentation mixture residue obtained after filtration was treated for 30 min by ultrasonic grinder, which promoted the cell fragmentation of microbial strain and the release of lipid in the cell. Centrifuge was used to separate the fermentation mixture, and the upper liquid was added to ether-petroleum ether, fully shaken and mixed, and left for 30 min to extract the lipid in the water phase. The lipid layer was sucked out, and then the ether-petroleum ether was steamed in a water bath (90 °C), and dried to constant weight in a constant temperature drying oven (105 °C). The lipid quality was obtained by using the difference method, and the amount of lipid produced per 100 g dry material was calculated. The fatty acid composition in the mixture of corn straw fermented by microbial strains was determined by gas chromatography (GC) analysis15.

Statistical analysis

A statistical analysis was carried out using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). The mean values and the standard error of the mean were calculated from the data obtained from three independent experiments. Student’s t test was used to eval‎uate the differences between the means of the test, and P < 0.05 was considered as significantly different.

Results

Effect of cellulase concentration on lipid production of engineered M. circinelloides strains from corn straw

The effect of cellulase concentration on lipid production of engineered M. circinelloides strains from corn straw was investigated as shown in Fig. 1. The results showed that when cellulase is not added, the amounts of lipids synthesized by the strains are little. With the increase of cellulase supplemented, lipid production gradually increased. When cellulase content reached 1.5%, the lipid yield reached the maximum. In view of the effect and cost savings of cellulase, the optimal amount of cellulase was determined to be 1.5%. When cellulase was added to 1.5%, the maximum lipid yields of the engineered strains were improved significantly by 58% for Mc-C2TD6 and 80% for Mc-C2PD6 compared with that of the control strain, respectively.

Figure 1

Lipid production of engineered M. circinelloides strains under corn straw solid medium containing different concentrations (0.0–2.0%, w/w) of cellulase. Strains were cultured in solid medium of corn straw during 192 h at 28 °C and pH 4.5 with an inoculum of 10%. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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Effect of inoculum amount on lipid production of engineered M. circinelloides strains from corn straw

The lipid yield of engineered M. circinelloides strains under corn straw solid medium with different inoculum concentrations (5.0–20.0%, w/w) were shown in Fig. 2. The results showed that with the increase of inoculation amount, lipid production gradually increased. When inoculation content reached 15%, the lipid yield reached the maximum. After that, the lipid yield did not increase significantly when the quantity of inoculation continued to increase. The optimal amount of inoculation was determined to be 15%. Similarly, under the optimal inoculation content, the lipid yields of the engineered strains were increased by 53% in Mc-C2TD6 and 72% in Mc-C2PD6 higher than that of the control strain, respectively.

Figure 2

Lipid production of engineered M. circinelloides strains under corn straw solid medium with different inoculum concentrations (5.0–20.0%, w/w). Strains were cultured in solid medium of corn straw during 192 h at 28 °C, pH 4.5 and supplemented with 1.5% cellulase. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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Lipid production of engineered M. circinelloides strains from corn straw during a temperature two-stage strategy

A two-stage temperature control strategy was adopted to investigate the lipid production of engineered M. circinelloides strains from corn straw under the condition of adding 1.5% cellulase and 15% inoculum. As shown in Fig. 3, the lipid accumulation patterns in different strains were similar: the lipid accumulation increased gradually with fermentation time until the highest lipid yield reached at 168 h, and then it slowed down and leveled off. Notably, the maximum lipid yields of engineered strains were 1.35 g for Mc-C2TD6 and 1.56 g for Mc-C2PD6 per 100 g dry medium, which were significantly enhanced by 1.5- and 1.8-fold, respectively, compared with the control strain. This indicated that the engineered M. circinelloides strains were more likely to cooperate with cellulase to synthesize lipids from corn straw.

Figure 3

Lipid production of engineered M. circinelloides strains under corn straw solid medium during a temperature two-stage strategy. Strains were cultured in solid medium of corn straw during 192 h at pH 4.5, inoculated at 15% and supplemented with 1.5% cellulase. The two-stage temperature control strategy was 32 °C for the first 48 h and 28 °C for the last 144 h. Values were mean of three independent fermentation experiments. Error bars represent the standard error of the mean.

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GLA production of engineered M. circinelloides strains from corn straw

The properties of fatty acids produced by engineered M. circinelloides strains using corn straw were shown in Table 2. The maximum GLA yields of engineered strains were 216 mg for Mc-C2TD6 and 274 mg for Mc-C2PD6 per 100 g dry medium. Compared with the control strain, the intracellular GLA yields of Mc-C2TD6 and Mc-C2PD6 were increased by 1.8- and 2.3-fold, respectively, which were consistent with our previous results on microcrystalline cellulose and carboxymethyl cellulose15,18. Therefore, the engineered M. circinelloides strains, especially for Mc-C2PD6, showed advantages in using corn straw to produce GLA.

Table 2 The fatty acid composition in engineered M. circinelloides strains cultured with corn straw.

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Discussion

Although microbial lipids become an important alternative resource for biodiesel production, and some functional lipids beneficial to people's health are also the main production objects of the food and pharmaceutical industry, high cost is still the main factor restricting their industrial production1,3,6,13. The use of cheap alternative raw materials (such as crop straw) is one of the important problems to solve the industrialization and scale of microbial lipids11,13. The recent researches focus on the strategies of raw material treatment, strain screening and optimization of fermentation to improve the microbial lipid production from cellulosic raw materials4,10,13. However, the adoption of efficient strains that could use cheap cellulosic feedstock is an important way to achieve high microbial lipid production.

It is believed that the oleaginous fungus M. circinelloides has the following specific advantages and potential over other oleaginous microorganisms in lipid production. Firstly, M. circinelloides was the first commercially lipid producing strain in the world, and its synthesized lipids are rich in GLA, which has the role of preventing inflammation, softening blood vessels, and lowering blood sugar and lipids16,19. Secondly, M. circinelloides can absorb and metabolize multiple sugars including glucose, xylose, and cellobiose, so it could make full use of cellulose and hemicellulose in crop straw, which has superiority in reducing production cost1,17,18. Thirdly, M. circinelloides has become a model organism to study the lipid mechanism in fungi with its successfully analyzed genome sequence and perfect gene manipulation system15,20,21. Finally, the ability of GLA synthesis from cellulose in M. circinelloides was enhanced through the genetic engineering, and the simultaneous conversion of cellulose-monosaccharide-lipids was realized in one step, which could not be achieved in other oleaginous microorganisms15,18. In view of this, the lipid production conditions from corn straw by engineered M. circinelloides strains Mc-C2TD6 and Mc-C2PD6 were investigated.

The complex structure of corn straw needs a series of pretreatments to be effectively degraded22,23. In this study, in order to improve the utilization efficiency of corn straw, cellulase was added in the fermentation process to promote the degradation of corn straw. The addition of cellulase is too little, resulting in poor degradation effect of corn straw. Excessive addition of cellulase can cause excessive cost. The amount of cellulase was added according to its production instructions, and the addition range is 0.5–2.0% (w/w), with 0.0% as a control. The results showed that the optimum concentration of cellulase was 1.5%. However, the lipid yield did not increase significantly when the quantity of cellulase continued to increase. The reason might be that cellulase can assist the engineered M. circinelloides strains to degrade cellulose in corn straw and produce reducing sugars, and meanwhile the stains can further use reducing sugars to synthesize lipids. Thus, with the increase of cellulase addition, the production of synthesized lipids also increased. When the amount of cellulase added reaches a certain value, the final lipid production does not increase or increases slightly due to the limited cellulose that can be degraded in the raw material. In addition, it could also be seen that with the assistance of cellulase, the engineered strains might be more efficient in the decomposition of corn straws, and the resultant lipid yields are much higher than that of the control strain (Mc-2075) and the wild strain (WT).

The amount of inoculation is an important factor affecting the growth and lipid production of engineered M. circinelloides strains from corn straw1. A larger amount of inoculation can shorten the time when mycelium propagation reaches its peak and make the formation of products arrive in advance24. However, too much inoculation will cause insufficient oxygen supply and affect product synthesis, and it is not economical. Generally, the inoculation amount of mold is usually 5–20% (w/w). Therefore, in order to determine the optimal inoculum, we set the inoculum range at dose of 5–20%. The results showed that the optimum inoculation amount was 15%.

Generally, the production of lipids from corn straw requires first degradation of corn straw into small molecules of available monosaccharides, and then these monosaccharides are converted into lipids by microbial fermentation1,25. In the early stage, the engineered M. circinelloides strains cooperated with cellulase to degrade corn straws, and then in the middle and late stages, the strains further used the decomposed small molecules of sugars to grow and synthesize lipids. However, the temperature of enzymatic hydrolysis of cellulose by cellulase was relatively high, while the temperature of growth and lipid accumulation of M. circinelloides was low. Thus, a two-stage temperature control strategy was adopted to investigate the lipid production of engineered M. circinelloides strains from corn straws under the condition of adding 1.5% cellulase and 15% inoculum. The maximum lipid and GLA yields of engineered strains were 1.56 g and 274 mg per 100 g dry medium (for Mc-C2PD6), which were significantly enhanced by 1.8- and 2.3-fold, respectively, compared with the control strain. These results indicated that the engineered M. circinelloides strains could cooperate with cellulase well to synthesize lipids and GLA from corn straw.

It should be noted that the pure biological treatment method of microbial and enzyme co-fermentation adopted in this study has incomparable advantages over traditional chemical treatment methods, such as non-toxic by-product inhibition, simple steps, and no environmental pollution, etc. However, it is undeniable that this study also has some limitations, such as long degradation process, incomplete degradation, few available monosaccharide and slow utilization of corn straw.

In conclusion, one of the current research hotspots of bioenergy is the production of biodiesel by microbial fermentation using lignocellulosic biomass such as crop straw. In the present study, the effects of cellulase concentration and inoculum amount on lipid production of engineered M. circinelloides strains from corn straw were analyzed. Furthermore, a two-stage temperature control strategy was developed to facilitate the lipid and GLA synthesis from corn straws by engineered M. circinelloides strains. This study laid a foundation for the direct microbial transformation from corn straw to functional GLA in one step, which might be helpful for reducing the production cost.

Data availability

All data generated or analysed during this study are included in this published article.

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