Hull Claire M, Warrilow Andrew G S, Rolley Nicola J, Price Claire L, Donnison Iain S, Kelly Diane E, Kelly Steven L
Institute of Life Science, Swansea University Medical School, Swansea, SA2 8PP Wales UK.
Institute of Biological, Environmental & Rural Sciences, Aberystwyth University, Gogerddan, Aberystwyth, Wales SY23 3EE UK.
Biotechnol Biofuels. 2017 Sep 29;10:226. doi: 10.1186/s13068-017-0904-z. eCollection 2017.
Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice.
Genes encoding the 11α-steroid hydroxylase enzymes from (11α-SH) and (CYP509C12) transformed into for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SH and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SH) or a 7:1 mixture of 11α-hydroxyprogesterone and 6β-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SH and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SH gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed.
The progesterone 11α-steroid hydroxylases from and , both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in . It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.
需要从可持续生物质来源生产生物乙醇，以限制对粮食生产的影响，并且在生物炼制方法中，从植物材料和微生物生物质中获得副产物是理想的。类固醇的真菌生物转化是最早允许生产皮质类固醇的工业生物转化之一。在这项工作中，在从多年生黑麦草汁生产生物乙醇之后，在实验室规模上证明了酵母生产皮质类固醇生产所需中间体的潜力。
将来自和的编码11α-类固醇羟化酶的基因（11α-SH和CYP509C12）转化到中，以便在p425TEF中进行异源组成型表达。两种重组酵母（AH22:p11α-SH和AH22:p509C12）均表现出高效的孕酮生物转化（在含有300μM孕酮的葡萄糖基本培养基上），产生11α-羟基孕酮作为唯一代谢物（AH22:p11α-SH）或11α-羟基孕酮和6β-羟基孕酮的7:1混合物（AH22:p509C12）。AH22:p11α-SH和AH22:p509C12的乙醇产率相当，导致葡萄糖向酒精的转化率≥75%。然后使用多年生黑麦草汁证明了生物乙醇的联产以及高效生产用于皮质类固醇制造的11-OH中间体。将11α-SH基因整合到酵母基因组中（AH22:11α-SHAoch+K），使用300μM孕酮时，11α-羟基孕酮的产率降低了36%，降至174μmol/L。然而，将孕酮浓度提高到955μM并优化生长条件，使11α-羟基孕酮的产量增加到592μmol/L产物形成。
来自和的孕酮11α-类固醇羟化酶，均为细胞色素P450超家族的单加氧酶，已在中功能性表达。似乎真菌中的这些活性与细胞色素P450的保守家族无关。酶的活性很重要，因为生物转化的特异性仅产生皮质类固醇生产所需的11-OH产物。所呈现的数据表明重组酵母如何能够在农村生物炼制过程中找到应用，在这些过程中，从新型原料联产增值产品（11α-羟基孕酮和乙醇）是一种新兴且有吸引力的可能性。
