Jin Hyunbin, Kim Sojeong, Lee Daeyeol, Ledesma-Amaro Rodrigo, Hahn Ji-Sook
School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, SW7 2AZ, UK.
Biotechnol Biofuels Bioprod. 2023 Oct 29;16(1):162. doi: 10.1186/s13068-023-02415-y.
Mycosporine-like amino acids (MAAs), including shinorine and porphyra-334, are gaining attention as safe natural sunscreens. The production of MAAs has been achieved in diverse microbial hosts, including Saccharomyces cerevisiae. While S. cerevisiae is the most extensively studied model yeast, the oleaginous yeast Yarrowia lipolytica has emerged as a promising candidate for the synthesis of valuable products. In this study, we explored the potential of Y. lipolytica as a host for producing MAAs, utilizing its advantages such as a robust pentose phosphate pathway flux and versatile carbon source utilization.
We produced MAAs in Y. lipolytica by introducing the MAA biosynthetic genes from cyanobacteria Nostoc punctiforme and Anabaena variabilis. These genes include mysA, mysB, and mysC responsible for producing mycosporine-glycine (MG) from sedoheptulose 7-phosphate (S7P). The two strains utilize different enzymes, D-Ala-D-Ala ligase homologue (MysD) in N. punctiforme and NRPS-like enzyme (MysE) in A. variabilis, for amino acid conjugation to MG. MysE specifically generated shinorine, a serine conjugate of MG, while MysD exhibited substrate promiscuity, yielding both shinorine and a small amount of porphyra-334, a threonine conjugate of MG. We enhanced MAAs production by selecting mysA, mysB, and mysC from A. variabilis and mysD from N. punctiforme based on their activities. We further improved production by strengthening promoters, increasing gene copies, and introducing the xylose utilization pathway. Co-utilization of xylose with glucose or glycerol increased MAAs production by boosting the S7P pool through the pentose phosphate pathway. Overexpressing GND1 and ZWF1, key genes in the pentose phosphate pathway, further enhanced MAAs production. The highest achieved MAAs level was 249.0 mg/L (207.4 mg/L shinorine and 41.6 mg/L of porphyra-334) in YP medium containing 10 g/L glucose and 10 g/L xylose.
Y. lipolytica was successfully engineered to produce MAAs, primarily shinorine. This achievement involved the introduction of MAA biosynthetic genes from cyanobacteria, establishing xylose utilizing pathway, and overexpressing the pentose phosphate pathway genes. These results highlight the potential of Y. lipolytica as a promising yeast chassis strain for MAAs production, notably attributed to its proficient expression of MysE enzyme, which remains non-functional in S. cerevisiae, and versatile utilization of carbon sources like glycerol.
包括肌醇六磷酸和紫菜-334在内的类菌孢素氨基酸（MAAs）作为安全的天然防晒剂正受到关注。MAAs已在多种微生物宿主中实现生产，包括酿酒酵母。虽然酿酒酵母是研究最广泛的模式酵母，但产油酵母解脂耶氏酵母已成为合成有价值产品的有前途的候选者。在本研究中，我们利用解脂耶氏酵母强大的磷酸戊糖途径通量和广泛的碳源利用等优势，探索其作为生产MAAs宿主的潜力。
我们通过引入来自点状念珠藻和多变鱼腥藻的MAAs生物合成基因，在解脂耶氏酵母中生产MAAs。这些基因包括负责从景天庚酮糖7-磷酸（S7P）生产肌醇六磷酸-甘氨酸（MG）的mysA、mysB和mysC。这两个菌株利用不同的酶，点状念珠藻中的D-丙氨酸-D-丙氨酸连接酶同源物（MysD）和多变鱼腥藻中的类非核糖体肽合成酶（MysE），将氨基酸与MG偶联。MysE特异性地产生肌醇六磷酸，MG的丝氨酸共轭物，而MysD表现出底物混杂性，产生肌醇六磷酸和少量紫菜-334，MG的苏氨酸共轭物。我们根据活性从多变鱼腥藻中选择mysA、mysB和mysC，从点状念珠藻中选择mysD，以提高MAAs产量。我们通过强化启动子、增加基因拷贝数和引入木糖利用途径进一步提高产量。木糖与葡萄糖或甘油的共同利用通过磷酸戊糖途径增加S7P库，从而提高MAAs产量。过表达磷酸戊糖途径中的关键基因GND1和ZWF1，进一步提高了MAAs产量。在含有10 g/L葡萄糖和10 g/L木糖的YP培养基中，达到的最高MAAs水平为249.0 mg/L（207.4 mg/L肌醇六磷酸和41.6 mg/L紫菜-334）。
解脂耶氏酵母已成功工程化以生产MAAs，主要是肌醇六磷酸。这一成果涉及引入来自蓝细菌的MAAs生物合成基因、建立木糖利用途径和过表达磷酸戊糖途径基因。这些结果突出了解脂耶氏酵母作为生产MAAs的有前途的酵母底盘菌株的潜力，特别是由于其MysE酶的高效表达，该酶在酿酒酵母中无功能，以及对甘油等碳源的广泛利用。
